The determination of carcinogenic activity in experimental compounds is a long and expensive process that negatively impacts many core industries and can threaten human health. Faster, more reliable methods that can be used to accurately determine human oncogenic activity are needed to rapidly identify environmental problems and reduce the reliance on animal models. The pleiotropic effects of carcinogens have made them difficult to detect using standard high-throughput assays that measure a single cellular change. A solution to this problem is to monitor several aspects of cell function simultaneously. Although these methods have the appropriate sensitivity, their implementation has been difficult due to long and complicated sample preparation procedures which decrease the throughput and dramatically increase the assay variability. The solution we propose in this grant is the multiplexed measurement of cellular responses in one sample using flow cytometry combined with cellular barcoding. In this system, multiple biosensors are encoded in a single sample to provide nine distinct measures of cellular responses to carcinogens. In addition, cell viability and cell cycle status are obtained for each sample, enabling multi-dimensional analysis of compound activity. This system benefits from the increased sensitivity of multiplexed measurement while retaining the precision of a homogeneous high-throughput assay. PUBLIC HEALTH RELEVANCE: Chemical carcinogens induce complex changes in cellular DNA and metabolism, making it difficult to identify carcinogens using single end-point assays. The platform described here uses high-content, multiplexed measurements of known oncogenic signaling pathways to improve the detection of carcinogens in clinical and environmental samples and provide information about their mechanism of action.